Staphylococcus aureus, an important human pathogen, expresses a multitude of secreted toxins (exotoxins) that can kill several different cell types, including erythrocytes, neutrophil granulocytes and other immune cells, as well as epithelial cells of the lung or skin. Moreover, most of these toxins activate immune cells and act as potent pro-inflammatory signals.
A prominent member of S. aureus cytotoxins is alpha hemolysin (Ha), which exerts cytolytic function on human lung epithelial and endothelial cells, lymphocytes and macrophages. It is also able to lyse rabbit red blood cells (RBCs) but much less toxic to human RBCs. Hla is considered to be the key virulence factor in S. aureus pneumonia pathogenesis and responsible for tissue damage via lysis of pulmonary epithelial cells and recruiting immune cells in mass quantity. The recruited phagocytic cells, mainly neutrophil granulocytes become targets for other cytotoxins produced by S. aureus during disease. The most potent toxins are the bi-component cytolysins, or leukocidins, formed by one S (slow eluted), and one F (fast eluted) component. The gamma hemolysin (Hlg) gene products—universally expressed by all S. aureus strains—can form two toxins: HlgAB and HlgCB (subunit B is the F-component), both are highly potent in killing human immune cells: PMNs, lymphocytes and macrophages. The former one is also a very potent hemolysin for human RBCs.
The Panton-Valentine Leukocidin (PVL), also called LukSF is the best characterized of the bi-component toxins. It is carried by phage derived genetic elements, and produced by approximately 5-10% of S. aureus strains isolated from patients, however, the rate of PVL-expressing strains is reported to be 50-93% in skin and soft tissue infections, depending on the type of disease (Lina, Clin Infect Dis, 1999:1128). LukED (LukD is the F-component) is a less potent leukocidin, but confirmed to be present in the majority of clinical S. aureus isolates (Shukla, J Clin Microbiol, 2010:3582). Initially, its role was implicated in skin infections only, but being the least characterized among the bi-component toxins, its contribution to other types of S. aureus infections can not be excluded. LukED has recently been reported to be involved in bloodstream infection in a murine model of S. aureus infection (Alonzo, Mol Microbiol, 2012:423). These two gene pairs share significant homology with each other (68-82% amino acid identity), while the recently identified leukocidin LukGH (LukG is the F-component) has a lower homology, with 33-40% identity (Ventura, PloS ONE, 2010:e11634; DuMont, Mol Microbiol, 2011:814).
The crystal structure of Hla, LukS, LukF, HlgA and HlgB have been determined, and revealed some structural homology, in spite of the low level of amino acid homology between Hla and the bi-component toxin subunits with 16-28% amino acid identity (Galdiero, Protein Sci, 2004:1503; Pedelacq, Structure, 1999:277; Menestrina, FEBS Letters, 2003:54). All these toxins form a ring-like structure formed by oligomerized subunits, leading to pore formation within the cell membrane and subsequent cytolysis. In case of Hla, the pore has been shown to be heptameric, but for the bi-component toxins, hexameric (Comai, Mol Microbiol, 2002:1251), heptameric and octameric (Yamashita, PNAS, 2011:17314) heterooligomers have been reported, leading to a debate within the scientific community (reviewed in detail by Kaneko, Biosci Biotechnol Biochem, 2004:981)
The different F- and S-components of this toxin family can form not only cognate pairs (these are: LukS-LukF, LukE-LukD, HlgC-HlgB, HlgA-HlgB and LukH-LukG), but also non-cognate pairs, many of those pairs reported by Gravet et al. (Gravet, FEBS Letters, 1998:202) and by Dalla Serra et al. for gamma hemolysins and LukS (Dalla Serra, J Chem Inf Model, 2005:1539) Due to the redundancy and promiscuous nature of this toxin family, inactivating one single component is unlikely to be effective to fight S. aureus infections. This notion is supported by observations reported in the literature when neutralization of a single bi-component toxin only partially affected the phenotype (e.g. Ventura, PloS ONE, 2010:e11634; Malachowa, PloS ONE, 2011:e18617). Animal studies showed a differential impact of the various bi-component toxins on the survival, depending on the model employed or the species used for in vivo experiments. The most prominent reduction in disease severity was observed when multiple toxins were deleted, e.g. as in a rabbit model of infection using a knock-out strain of S. aureus where the agr quorum sensing system, a global regulator of toxin expression was inactivated (Kobayashi, J Infect Dis, 2011:204). Therefore, it is expected that antibody cocktails neutralizing more toxins offer a significant advantage over mAbs against single toxins. However, monoclonal antibody (mAb) cocktails comprising of more than three components are challenging to be developed.
The likelihood of finding single antibodies that cross-react between alpha hemolysin and any of the bi-component toxins was considered to be low, based on the low (<30%) sequence homology between Hla and bi-component toxins. The chance of finding single antibodies cross-reactive among S- and F-components is expected to be higher, due to the higher level of sequence homology, with the exception of LukGH. It has been described that hyperimmune serum from animals immunized with LukS can recognize HlgC, however, this is due to the presence of different specificities in the polyclonal serum. Laventie et al. (Laventie, PNAS, 2011:16404) described a bi-specific neutralizing antibody against LukS and HlgC. This type of antibody however cannot form avid interaction due to the single binding sites on the cognate antigen. The cross-reactivity of such bi-specific mAbs is normally limited to two specificities, i.e. it does not offer the potential of broad cross-neutralization. In summary, no cross-reactive mAbs against different bi-component S. aureus toxins or against alpha hemolysin and any of the bi-component toxins have been reported to date.